Cytochrome c oxidase (COX) deficiency is the most frequent cause of mitochondrial neuromyopathiesin humans. Patients afflicted with these diseases present heterogeneous clinical phenotypes, including Leigh syndrome, muscle weakness and encephalomyopathy.A complete understanding of COX biogenesis is essential for elucidating the molecular basis underlying this group of diseases. The main objective of the proposed research is to use the yeast Saccharomycescerevisiae as a model to investigate COXassembly in wild type cells and in cells with mutations in evolutionary conservedassembly factors. Several specific aims will be pursued. 1) We have recently reported that Shylp, the yeast homologue of humanSurMp, responsible for most cases of Leigh's syndrome,catalyzes the formation of a COX assembly intermediate involving Coxlp, a mitochondrially encoded catalytic subunit of COX. The role of Shylp in expression of Coxlp will be studied. 2) More recent evidence indicates that this intermediate regulates Cox1 pexpression in a process involving other COX metabolism factors, such as Mss51p and Cox14p.The mechanisms by which these proteins regulate COX expression will be studied. Appropriately tagged Mss51p and Cox14p will be purified from over-expressing yeast cells. The availability of purified proteins will permit hypotheses concerning their activities to be tested directly. 3) The proteins involved in regulation of Coxlp synthesis by COX assembly are likely to interact transiently or permanently amongthem to perform their functions. The nature of these interactions will be characterized. In summary,the yeast system will be explored as a means of deciphering the general principles operating in the assembly of a complex membrane enzyme composed of subunit polypeptides derived from two spatially separated genetic sources. The yeast paradigm will be exploited by biochemical and genetic meansto gain a complete understanding of the function of Shylp and therefore of Surflp as well, and to clarifythe molecular basis of human COX deficiencies.